Modified cytokines for use in cancer therapy

ABSTRACT

Cytokine derivatives capable of homing the tumoral vessels and the antigen presenting cells and the use thereof as antitumoral agents.

FIELD OF THE INVENTION

[0001] The present invention refers to modified cytokines for use in thetreatment of cancer. More particularly, the invention refers tocytokines derivatives capable of “homing” tumor vessels and antigenpresenting cells. The present invention also relates to synergisticcombinations which include a modified cytokine.

BACKGROUND TO THE INVENTION

[0002] The antitumoral activity of some cytokines is well known anddescribed. Some cytokines have already been used therapeutically also inhumans (29). For example, such cytokines as interleukine-2 (IL-2) andinterferon α(IFNα) have shown positive antitumoral activity in patientswith different types of tumors, such as kidney metastatic carcinoma,hairy cell leukemia, Kaposi sarcoma, melanoma, multiple mieloma, and thelike. Other cytokines like IFNβ, the Tumor Necrosis Factor (TNF) α,TNFβ, IL-1, 4, 6, 12, 15 and the Colony Stimulating Factors (CFSs) haveshown a certain antitumoral activity on some types of tumors andtherefore are the object of further studies.

[0003] In general, the therapeutic use of cytokines is strongly limitedby their systemic toxicity. TNF, for example, was originally discoveredfor its capacity of inducing the hemorrhagic necrosis of some tumors(1), and for its in vitro cytotoxic effect on different tumoral lines(2), but it subsequently proved to have strong pro-inflammatoryactivity, which can, in case of overproduction conditions, dangerouslyaffect the human body (3).

[0004] As the systemic toxicity is a fundamental problem with the use ofpharmacologically active amounts of cytokines in humans, novelderivatives and therapeutic strategies are now under evaluation, aimedat reducing the toxic effects of this class of biological effectorswhile keeping their therapeutic efficacy.

[0005] Some novel approaches are directed to:

[0006] a) the development of fusion proteins which can deliver TNF intothe tumor and increase the local concentration. For example, the fusionproteins consisting of TNF and tumor specific-antibodies have beenproduced (4);

[0007] b) the development of TNF mutants which maintain the antitumoralactivity and have a reduced systemic toxicity. Accordingly, mutants ableof selectively recognizing only one receptor (p55 or p75) have beenalready prepared (5);

[0008] c) the use of anti-TNF antibodies able to reduce some toxiceffects of TNF without compromising its antitumoral activity. Suchantibodies have been already described in literature (30);

[0009] d) the use of TNF derivatives with a higher half-life (forexample TNF conjugated with polyethylene glycol).

[0010] The preparation of TNF derivatives capable of selectivelytargeting the tumoral sites has been recently reported. For example, afusion protein has been described, obtained by fusing the gene of theheavy chain of an anti-transferrin receptor mAb and the TNF gene (4), ora fusion protein of TNF with the “hinge” region of a monoclonal antibodyagainst the tumor-associated TAG72 antigen (6), or a Fv-TNF fusionprotein (6).

[0011] EP 251 494 discloses a system for administering a diagnostic ortherapeutic agent, which comprises: an antibody conjugated with avidinor streptavidin, an agent capable of complexing the conjugated antibodyand a compound consisting of the diagnostic or therapeutic agentconjugated with biotin, which are administered sequentially andadequately delayed, so as to allow the localization of the therapeuticor diagnostic agent through the biotin-streptavidin interaction on thetarget cell recognized by the antibody. The described therapeutic ordiagnostic agents comprise metal chelates, in particular chelates ofradionuclides and low molecular weight antitumoral agents such ascis-platinum, doxorubicin, etc.

[0012] EP 496 074 discloses a method which provides the sequentialadministration of a biotinylated antibody, avidin or streptavidin and abiotinylated diagnostic or therapeutic agent. Although cytotoxic agentslike ricin are generically mentioned, the application relative toradiolabelled compounds is mostly disclosed.

[0013] WO 95/15979 discloses a method for localizing highly toxic agentson cellular targets, based on the administration of a first conjugatecomprising the specific target molecule conjugated with a ligand or ananti-ligand followed by the administration of a second conjugateconsisting of the toxic agent bound to an anti-ligand or to the ligand.

[0014] WO098/10795 discloses tumor homing molecules including peptidescontaining the amino acid sequence NGR. No use of the peptide to targeta cytokine to a tumor is described.

[0015] WO 99/13329 discloses a method for targeting a molecule totumoral angiogenic vessels, based on the conjugation of said moleculewith ligands of NGR receptors. A number of molecules have been suggestedas possible candidates, but doxorubicin only is specifically described.No use of ligands of NGR receptors as cytokines vehicles to induceimmuno responses is disclosed.

SUMMARY OF THE INVENTION

[0016] It has now surprisingly been found that the therapeutic index ofcertain cytokines can be remarkably improved and their immunotherapeuticproperties can be enhanced by coupling with a ligand of aminopeptidase-Nreceptor (CD13). CD13 is a trans-membrane glycoprotein of 150 kDa highlyconserved in various species. It is expressed on normal cells as well asin myeloid tumor lines, in the angiogenic endothelium and is someepithelia. CD13 receptor is usually identified as “NGR” receptor, inthat its peptide ligands share the amino acidic “NGR” motif. We havealso surprisingly found that TNF coupled with a ligand of CD13 receptorand IFNγ act synergistically so that effective anti-tumor activity maybe seen upon co-administration at dosages which are below the effectivedoses individually. In addition, we have found that the anti-tumoractivity of a combination of the modified TNF and another anti-tumoragent, such as doxorubicin, is increased by administration of IFNγ.

STATEMENTS OF THE INVENTION

[0017] According to a first aspect, the invention provides a conjugationproduct of a cytokine selected from TNF and IFNγ and a ligand of CD13receptor.

[0018] According to another aspect of the present invention there isprovided a pharmaceutical composition comprising an effective amount ofa conjugation product of TNF and a ligand of the CD13 receptor, and aneffective amount of IFNγ.

DETAILED DESCRIPTION OF THE INVENTION

[0019] Various preferred features and embodiments of the presentinvention will now be described by way of non-limiting example.

[0020] Said ligand of CD13 receptor can be an antibody or a fragmentthereof such as Fab, Fv, single-chain Fv, a peptide or apeptido-mimetic, namely a peptido-like molecule capable to bind the CD13receptor, optionally containing modified, not naturally occurring aminoacids.

[0021] CD13 is a trans-membrane glycoprotein of 150 kDa highly conservedin various species. It is expressed on normal cells as well as inmyeloid tumor lines, in the angiogenic endothelium and is someepithelia. CD13 receptor is usually identified as “NGR” receptor. Theligands may be natural or synthetic. The term “ligand” also refers to achemically modified ligand. The one or more binding domains of theligand may consist of, for example, a natural ligand for the receptor,or a fragment of a natural ligand which retains binding affinity for thereceptor. Synthetic ligands include the designer ligands. As usedherein, the term means “designer ligands” refers to agents which arelikely to bind to the receptor based on their three dimensional shapecompared to that of the receptor.

[0022] The ligand is preferably a straight or cyclic peptide comprisingthe NGR motif, such as CNGRCVSGCAGRC, NGRAHA, GNGRG, cycloCVLNGRMEC orcycloCNGRC, or, more preferably, the peptide CNGRC. Such ligands aredescribed in WO98/10795 which is herein incorporated by reference.Methods of identifying ligands of CD13 receptor are disclosed inWO99/13329 which is herein incorporated by reference.

[0023] In one embodiment, the method of screening for an agent capableof binding to a CD13 receptor, the method comprising contacting the cellsurface molecule with an agent and determining if said agent binds tosaid cell surface molecule.

[0024] As used herein, the term “agent” includes, but is not limited to,a compound, such as a test compound, which may be obtainable from orproduced by any suitable source, whether natural or not. The agent maybe designed or obtained from a library of compounds which may comprisepeptides, as well as other compounds, such as small organic moleculesand particularly new lead compounds. By way of example, the agent may bea natural substance, a biological macromolecule, or an extract made frombiological materials such as bacteria, fungi, or animal (particularlymammalian) cells or tissues, an organic or an inorganic molecule, asynthetic test compound, a semi-synthetic test compound, a structural orfunctional mimetic, a peptide, a peptidomimetics, a derivatised testcompound, a peptide cleaved from a whole protein, or a peptidessynthesised synthetically (such as, by way of example, either using apeptide synthesizer) or by recombinant techniques or combinationsthereof, a recombinant test compound, a natural or a non-natural testcompound, a fusion protein or equivalent thereof and mutants,derivatives or combinations thereof.

[0025] The agent can be an amino acid sequence or a chemical derivativethereof. The substance may even be an organic compound or otherchemical.

[0026] As used herein the term “peptidomimetic” is used broadly to referto a peptide-like molecule that has the binding activity of the CD13ligand.

[0027] Alternatively, the ligand may be derived from heavy and lightchain sequences from an immunoglobulin (Ig) variable region. Such avariable region may be derived from a natural human antibody or anantibody from another species such as a rodent antibody. Alternativelythe variable region may be derived from an engineered antibody such as ahumanised antibody or from a phage display library from an immunised ora non-immunised animal or a mutagenised phage-display library. As asecond alternative, the variable region may be derived from asingle-chain variable fragment (scFv). The ligand may contain othersequences to achieve multimerisation or to act as spacers between thebinding domains or which result from the insertion of restriction sitesin the genes encoding the ligand, including Ig hinge sequences or novelspacers and engineered linker sequences.

[0028] The ligand may comprise, in addition to one or moreimmunoglobulin variable regions, all or part of an Ig heavy chainconstant region and so may comprise a natural whole Ig, an engineeredIg, an engineered Ig-like molecule, a single-chain Ig or a single-chainIg like molecule. Alternatively, or in addition, the BP may contain oneor more domains from another protein such as a toxin.

[0029] As used herein, an “antibody” refers to a protein consisting ofone or more polypeptides substantially encoded by immunoglobulin genesor fragments of immunoglobulin genes. Antibodies may exist as intactimmunoglobulins or as a number of fragments, including thosewell-characterised fragments produced by digestion with variouspeptidases. While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate thatantibody fragments may be synthesised de novo either chemically or byutilising recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesised de novo usingrecombinant DNA methodologies. Antibody fragments encompassed by the useof the term “antibodies” include, but are not limited to, Fab, Fab′, F(ab′) 2, scFv, Fv, dsFv diabody, and Fd fragments.

[0030] If polyclonal antibodies are desired, a selected mammal (e.g.,mouse, rabbit, goat, horse, etc.) is immunised with an immunogenicpolypeptide bearing an epitope(s). Serum from the immunised animal iscollected and treated according to known procedures. If serum containingpolyclonal antibodies to an epitope contains antibodies to otherantigens, the polyclonal antibodies can be purified by immunoaffinitychromatography. Techniques for producing and processing polyclonalantisera are known in the art. In order that such antibodies may bemade, the invention also provides polypeptides of the invention orfragments thereof haptenised to another polypeptide for use asimmunogens in animals or humans.

[0031] Monoclonal antibodies directed against binding cell surfaceepitopes in the polypeptides can also be readily produced by one skilledin the art. The general methodology for making monoclonal antibodies byhybridomas is well known. Immortal antibody-producing cell lines can becreated by cell fusion, and also by other techniques such as directtransformation of B lymphocytes with oncogenic DNA, or transfection withEpstein-Barr virus. Panels of monoclonal antibodies produced againstepitopes can be screened for various properties; i.e., for isotype andepitope affinity.

[0032] An alternative technique involves screening phage displaylibraries where, for example the phage express scFv fragments on thesurface of their coat with a large variety of complementaritydetermining regions (CDRs). This technique is well known in the art.

[0033] For the purposes of this invention, the term “antibody”, unlessspecified to the contrary, includes fragments of whole antibodies whichretain their binding activity for a target antigen. As mentioned abovesuch fragments include Fv, F(ab′) and F(ab′)₂ fragments, as well assingle chain antibodies (scFv). Furthermore, the antibodies andfragments thereof may be humanised antibodies, for example as describedin EP-A-239400.

[0034] The term “peptide” as used herein includes polypeptides andproteins. The term “polypeptide” includes single-chain polypeptidemolecules as well as multiple-polypeptide complexes where individualconstituent polypeptides are linked by covalent or non-covalent means.The term “polypeptide” includes peptides of two or more amino acids inlength, typically having more than 5, 10 or 20 amino acids.

[0035] It will be understood that polypeptide sequences for use in theinvention are not limited to the particular sequences or fragmentsthereof but also include homologous sequences obtained from any source,for example related viral/bacterial proteins, cellular homologues andsynthetic peptides, as well as variants or derivatives thereof.Polypeptide sequences of the present invention also include polypeptidesencoded by polynucleotides of the present invention.

[0036] The terms “variant” or “derivative” in relation to the amino acidsequences of the present invention includes any substitution of,variation of, modification of, replacement of, deletion of or additionof one (or more) amino acids from or to the sequence providing theresultant amino acid sequence preferably has targeting activity,preferably having at least 25 to 50% of the activity as the polypeptidespresented in the sequence listings, more preferably at leastsubstantially the same activity.

[0037] Thus, sequences may be modified for use in the present invention.Typically, modifications are made that maintain the activity of thesequence. Thus, in one embodiment, amino acid substitutions may be made,for example from 1, 2 or 3 to 10, 20 or 30 substitutions provided thatthe modified sequence retains at least about 25 to 50% of, orsubstantially the same activity. However, in an alternative embodiment,modifications to the amino acid sequences of a polypeptide of theinvention may be made intentionally to reduce the biological activity ofthe polypeptide. For example truncated polypeptides that remain capableof binding to target molecule but lack functional effector domains maybe useful.

[0038] In general, preferably less than 20%, 10% or 5% of the amino acidresidues of a variant or derivative are altered as compared with thecorresponding region depicted in the sequence listings.

[0039] Amino acid substitutions may include the use of non-naturallyoccurring analogues, for example to increase blood plasma half-life of atherapeutically administered polypeptide (see below for further detailson the production of peptide derivatives for use in therapy).

[0040] Conservative substitutions may be made, for example according tothe Table below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other: ALIPHATIC Non-polar G A P I L V Polar-uncharged C S T M N QPolar-charged D E K R AROMATIC H F W Y

[0041] Polypeptides of the invention also include fragments of the abovementioned polypeptides and variants thereof, including fragments of thesequences. Preferred fragments include those which include an epitope orbinding domain. Suitable fragments will be at least about 5, e.g. 10,12, 15 or 20 amino acids in length. They may also be less than 200, 100or 50 amino acids in length. Polypeptide fragments of the proteins andallelic and species variants thereof may contain one or more (e.g. 2, 3,5, or 10) substitutions, deletions or insertions, including conservedsubstitutions. Where substitutions, deletion and/or insertions have beenmade, for example by means of recombinant technology, preferably lessthan 20%, 10% or 5% of the amino acid residues depicted in the sequencelistings are altered.

[0042] Polypeptides and conjugates of the invention are typically madeby recombinant means, for example as described below. However they mayalso be made by synthetic means using techniques well known to skilledpersons such as solid phase synthesis. Various techniques for chemicalsynthesising peptides are reviewed by Borgia and Fields, 2000, TibTech18: 243-251 and described in detail in the references contained therein.

[0043] The peptide can be coupled directly to the cytokine or indirectlythrough a spacer, which can be a single amino acid, an amino acidsequence or an organic residue, such as6-aminocapryl-N-hydroxysuccinimide. The coupling procedures are known tothose skilled in the art and comprise genetic engineering or chemicalsynthesis techniques.

[0044] The peptide ligand preferably is linked to the cytokineN-terminus thus minimizing any interference in the binding of themodified cytokine to its receptor. Alternatively, the peptide can belinked to amino acid residues which are amido- or carboxylic-bondsacceptors, naturally occurring on the molecule or artificially insertedwith genetic engineering techniques. The modified cytokine is preferablyprepared by use of a cDNA comprising a 5′-contiguous sequence encodingthe peptide.

[0045] According to a preferred embodiment, there is provided aconjugation product between TNF and the CNGRC sequence. More preferably,the amino-terminal of TNF is linked to the CNGRC peptide through thespacer G (glycine).

[0046] The resulting product (NGR-TNF), proved to be more active thanTNF on RMA-T lymphoma animal models. Furthermore, animals treated withNGR-TNF were able to reject further tumorigenic doses of RMA-T or RMAcells. The increase in the antitumoral activity, as compared with normalTNF, could be observed in immunocompetent animals but not inimmunodeficient animals. This indicates that the increase in theantitumoral activity of TNF conjugated with “NGR” peptides is due to anenhanced immune response rather than to a direct cytotoxic activity ofthe conjugate.

[0047] It has also been demonstrated that the in vivo immune effectsinduced by NGR-TNF are directly related to the CD13 receptor. It has,for example, been observed that antibody against the CD13 receptor aswell as the GNGRC ligand compete with NGR-TNF in vivo, thus suggesting amechanism of receptor targeting by NGR-TNF.

[0048] The therapeutic index of the TNF/CD13 ligand conjugates can befurther improved by using a mutant form of TNF capable of selectivelybinding to one of the two TNF receptors, p75TNFR and p55TNFR. Said TNFmutant can be obtained by site-directed mutagenesis (5; 7).

[0049] The pharmacokinetic of the modified cytokines according to theinvention can be improved by preparing polyethylene glycol derivatives,which allow to extend the plasmatic half-life of the cytokinesthemselves.

[0050] A further embodiment of the invention is provided by bifunctionalderivatives in which the cytokines modified with the CD13 ligand areconjugated with antibodies, or their fragments, against tumoral antigensor other tumor angiogenic markers, e.g. αv integrins, metalloproteasesor the vascular growth factor, or antibodies or fragments thereofdirected against components of the extracellular matrix, such asanti-tenascin antibodies or anti-fibronectin EDB domain. The preparationof a fusion product between TNF and the hinge region of a mAb againstthe tumor-associated TAG72 antigen expressed by gastric and ovarianadenocarcinoma has recently been reported (6).

[0051] A further embodiment of the invention is provided by the tumoralpre-targeting with the biotin/avidin system. According to this approach,a ternary complex is obtained on the tumoral antigenic site, atdifferent stages, which is formed by 1) biotinylated mAb, 2) avidin (orstreptavidin) and 3) bivalent cytokine modified with the CD13 ligand andbiotin. A number of papers proved that the pre-targeting approach,compared with conventional targeting with immunoconjugates, can actuallyincrease the ratio of active molecule homed at the target to free activemolecule, thus reducing the treatment toxicity (11, 10, 9, 8). Thisapproach produced favorable results with biotinylated TNF, which wascapable of inducing cytotoxicity in vitro and decreasing the tumor cellsgrowth under conditions in which normal TNF was inactive (14, 26). Thepre-targeting approach can also be carried out with a two-phaseprocedure by using a bispecific antibody which at the same time bindsthe tumoral antigen and the modified cytokine. The use of a bispecificantibody directed against a carcinoembryonic antigen and TNF hasrecently been described as a means for TNF tumoral pre-targeting (31).

[0052] According to a further embodiment, the invention comprises a TNFmolecule conjugated to both a CD13 ligand and an antibody, or a fragmentthereof (directly or indirectly via a biotin-avidin bridge), ondifferent TNF subunits, where the antibody or its fragments are directedagainst an antigen expressed on tumor cells or other components of thetumor stroma, e.g. tenascin and fibronectin EDB domain. This results ina further improvement of the tumor homing properties of the modifiedcytokine and in the slow release of the latter in the tumormicroenvironment through trimer-monomer-trimer transitions. As shown inprevious works, in fact, the modified subunits of TNF conjugates candissociate from the targeting complexes and reassociate so as to formunmodified trimeric TNF molecules, which then diffuse in the tumormicroenvironment. The release of bioactive TNF has been shown to occurwithin 24-48 hours after targeting (21).

[0053] Peptides of the present invention may be administeredtherapeutically to patients. It is preferred to use peptides that do notconsisting solely of naturally-occurring amino acids but which have beenmodified, for example to reduce immunogenicity, to increase circulatoryhalf-life in the body of the patient, to enhance bioavailability and/orto enhance efficacy and/or specificity.

[0054] A number of approaches have been used to modify peptides fortherapeutic application. One approach is to link the peptides orproteins to a variety of polymers, such as polyethylene glycol (PEG) andpolypropylene glycol (PPG)—see for example U.S. Pat. Nos. 5,091,176,5,214,131 and U.S. Pat. No. 5,264,209.

[0055] Replacement of naturally-occurring amino acids with a variety ofuncoded or modified amino acids such as D-amino acids and N-methyl aminoacids may also be used to modify peptides

[0056] Another approach is to use bifunctional crosslinkers, such asN-succinimidyl 3-(2 pyridyldithio) propionate, succinimidyl 6-[3-(2pyridyldithio) propionamido] hexanoate, and sulfosuccinimidyl 6-[3-(2pyridyldithio) propionamido]hexanoate (see U.S. Pat. No. 5,580,853).

[0057] It may be desirable to use derivatives of the peptides of theinvention which are conformationally constrained. Conformationalconstraint refers to the stability and preferred conformation of thethree-dimensional shape assumed by a peptide. Conformational constraintsinclude local constraints, involving restricting the conformationalmobility of a single residue in a peptide; regional constraints,involving restricting the conformational mobility of a group ofresidues, which residues may form some secondary structural unit; andglobal constraints, involving the entire peptide structure.

[0058] The active conformation of the peptide may be stabilised by acovalent modification, such as cyclization or by incorporation ofgamma-lactam or other types of bridges. For example, side chains can becyclized to the backbone so as create a L-gamma-lactam moiety on eachside of the interaction site. See, generally, Hruby et al.,“Applications of Synthetic Peptides,” in Synthetic Peptides: A User'sGuide: 259-345 (W. H. Freeman & Co. 1992). Cyclization also can beachieved, for example, by formation of cysteine bridges, coupling ofamino and carboxy terminal groups of respective terminal amino acids, orcoupling of the amino group of a Lys residue or a related homolog with acarboxy group of Asp, Glu or a related homolog. Coupling of thealpha-amino group of a polypeptide with the epsilon-amino group of alysine residue, using iodoacetic anhydride, can be also undertaken. SeeWood and Wetzel, 1992, Int'l J. Peptide Protein Res. 39: 533-39.

[0059] Another approach described in U.S. Pat. No. 5,891,418 is toinclude a metal-ion complexing backbone in the peptide structure.Typically, the preferred metal-peptide backbone is based on therequisite number of particular coordinating groups required by thecoordination sphere of a given complexing metal ion. In general, most ofthe metal ions that may prove useful have a coordination number of fourto six. The nature of the coordinating groups in the peptide chainincludes nitrogen atoms with amine, amide, imidazole, or guanidinofunctionalities; sulfur atoms of thiols or disulfides; and oxygen atomsof hydroxy, phenolic, carbonyl, or carboxyl functionalities. Inaddition, the peptide chain or individual amino acids can be chemicallyaltered to include a coordinating group, such as for example oxime,hydrazino, sulfhydryl, phosphate, cyano, pyridino, piperidino, ormorpholino. The peptide construct can be either linear or cyclic,however a linear construct is typically preferred. One example of asmall linear peptide is Gly-Gly-Gly-Gly which has four nitrogens (an N₄complexation system) in the back bone that can complex to a metal ionwith a coordination number of four.

[0060] A further technique for improving the properties of therapeuticpeptides is to use non-peptide peptidomimetics. A wide variety of usefultechniques may be used to elucidating the precise structure of apeptide. These techniques include amino acid sequencing, x-raycrystallography, mass spectroscopy, nuclear magnetic resonancespectroscopy, computer-assisted molecular modelling, peptide mapping,and combinations thereof. Structural analysis of a peptide generallyprovides a large body of data which comprise the amino acid sequence ofthe peptide as well as the three-dimensional positioning of its atomiccomponents. From this information, non-peptide peptidomimetics may bedesigned that have the required chemical functionalities for therapeuticactivity but are more stable, for example less susceptible to biologicaldegradation. An example of this approach is provided in U.S. Pat. No.5,811,512.

[0061] Techniques for chemically synthesising therapeutic peptides ofthe invention are described in the above references and also reviewed byBorgia and Fields, 2000, TibTech 18: 243-251 and described in detail inthe references contained therein.

[0062] For use in therapy, the modified cytokines of the invention willbe suitably formulated in pharmaceutical preparations for the oral orparenteral administration. Formulations for the parenteraladministration are preferred, and they comprise injectable solutions orsuspensions and liquids for infusions. For the preparation of theparenteral forms, an effective amount of the active ingredient will bedissolved or suspended in a sterile carrier, optionally addingexcipients such as solubilizers, isotonicity agents, preservatives,stabilizers, emulsifiers or dispersing agents, and it will besubsequently distributed in sealed vials or ampoules.

[0063] In more detail, conjugates of the invention, includingpolypeptides and polynucleotides, may preferably be combined withvarious components to produce compositions of the invention. Preferablythe compositions are combined with a pharmaceutically acceptablecarrier, diluent or excipient to produce a pharmaceutical composition(which may be for human or animal use). Suitable carriers and diluentsinclude isotonic saline solutions, for example phosphate-bufferedsaline. Details of excipients may be found in The Handbook ofPharmaceutical Excipients, 2nd Edn, Eds Wade & Weller, AmericanPharmaceutical Association. The composition of the invention may beadministered by direct injection. The composition may be formulated forparenteral, intramuscular, intravenous, subcutaneous, intraocular, oralor transdermal administration.

[0064] The composition may be formulated such that administration daily,weekly or monthly will provide the desired daily dosage. It will beappreciated that the composition may be conveniently formulated foradministrated less frequently, such as every 2, 4, 6, 8, 10 or 12 hours.

[0065] Polynucleotides/vectors encoding polypeptide components may beadministered directly as a naked nucleic acid construct, preferablyfurther comprising flanking sequences homologous to the host cellgenome.

[0066] Uptake of naked nucleic acid constructs by mammalian cells isenhanced by several known transfection techniques for example thoseincluding the use of transfection agents. Example of these agentsinclude cationic agents (for example calcium phosphate and DEAE-dextran)and lipofectants (for example lipofectam™ and transfectam™). Typically,nucleic acid constructs are mixed with the transfection agent to producea composition.

[0067] Preferably the polynucleotide or vector of the invention iscombined with a pharmaceutically acceptable carrier or diluent toproduce a pharmaceutical composition. Suitable carriers and diluentsinclude isotonic saline solutions, for example phosphate-bufferedsaline. The composition may be formulated for parenteral, intramuscular,intravenous, subcutaneous, intraocular or transdermal administration.

[0068] The routes of administration and dosage regimens described areintended only as a guide since a skilled practitioner will be able todetermine readily the optimum route of administration and dosageregimens for any particular patient and condition.

[0069] The preparation of cytokines in form of liposomes can improve thebiological activity thereof. It has, in fact, been observed thatacylation of the TNF amino groups induces an increase in itshydrophobicity without loss of biological activity in vitro.Furthermore, it has been reported that TNF bound to lipids hasunaffected cytotoxicity in vitro, immunomodulating effects and reducedtoxicity in vivo (12, 13).

[0070] The maximum tolerated dose of bolus TNF in humans is 218-410μg/m² (32) about 10-fold lower than the effective dose in animals. Basedon data from murine models it is believed that an at least 10 timeshigher dose is necessary to achieve anti-tumor effects in humans (15).In the first clinical study on hyperthermic isolated-limb perfusion,high response rates were obtained with the unique dose of 4 mg of TNF incombination with melphalan and interferon γ (16). Other works showedthat interferon γ can be omitted and that even lower doses of TNF can besufficient to induce a therapeutic response (17, 18). As the twocytokines exert synergistic effects on endothelial cells, theircombined, selective targeting thereon is likely to result in strongeranti-tumor activity thus allowing to overcome the problems of systemictoxicity usually encountered in cancer therapy with the same cytokinesused in combination. Furthermore, it is known that TNF can decrease thebarrier function of the endothelial lining vessels, thus increasingtheir permeability to macromolecules. Taking advantage of the lowertoxicity of treatment with the modified TNF molecules according to theinvention, and of their tumor vessels homing properties, an alternativeapplication is their use to increase the permeability of tumor vesselsto other compounds, either for therapeutic or diagnostic purposes. Forinstance the modified TNF can be used to increase the tumor uptake ofradiolabelled antibodies or hormones (tumor-imaging compounds) inradioimmunoscintigraphy or radioimmunotherapy of tumors. Alternatively,the uptake of chemotherapeutic drugs, immunotoxins, liposomes carryingdrugs or genes, or other anticancer drugs could also be increased, sothat their antitumor effects are enhanced.

[0071] Accordingly, the cytokines of the invention can be used incombined, separated or sequential preparations, also with otherdiagnostic or therapeutic substances, in the treatment or in thediagnosis of cancer.

[0072] Another aspect of the present invention relates to the use of acombination of the modified TNF, and IFNγ. This combination can be usedin combined, separated or sequential preparations. Advantageously thecombination is also with other diagnostic or therapeutic substances, inthe treatment or in the diagnosis of cancer, such as doxorubicin andmephalan. Thus the present invention provides a pharmaceuticalcomposition comprising a combination of the modified TNF and IFNγ, andoptionally another tumor-diagnostic or anti-tumor therapeutic substance.Again, this combination can be used in combined, separated or sequentialpreparations.

[0073] In our patent application Ser. No. GB 02098960, we found targeteddelivery of picogram doses of cytokines enhances the penetration ofchemotherapeutic drugs, providing a novel and surprising strategy forincreasing the therapeutic index of chemotherapeutic drugs. Patentapplication Ser. No. GB 02098960 is hereby incorporated by reference inits entirety. In more detail, we have found that delivery of very lowdoses of cytokines to tumors and the tumor-associated environmentincluding tumor vasculature represents a new approach to avoidingnegative feedback mechanisms and to preserve its ability to alterdrug-penetration barriers.

[0074] The composition of the present invention may be formulated forparenteral, intramuscular, intravenous, subcutaneous, intraocular, oralor transdermal administration. In one embodiment of this aspect of thepresent invention, a conjugate of the present invention may beadministered at a dose of from in the range of 0.5 to 500 ng/kg,preferably in the range of 1 to 50 ng/kg, more preferably in the rangeof 5 to 15 ng/kg.

[0075] In an alternative embodiment of this aspect of the inventionthere is provided a pharmaceutical composition comprising a conjugate ofthe present invention in combination with IFNγ, wherein the conjugate ispresent in an amount such that the conjugate or a metabolite thereof isprovided to the blood plasma of the subject to be treated in an amountof no greater than about 35,000 ng/day, preferably about 3,500 ng/day,more preferably about 1,000 ng/day.

[0076] The above dosage relate to a dosage for a 70 kg subject. A personskilled in the art would readily be able to modify the recited dosagefor a subject having as mass other than 70 kg.

[0077] The routes of administration and dosage regimens described areintended only as a guide since a skilled practitioner will be able todetermine readily the optimum route of administration and dosageregimens for any particular patient and condition.

[0078] Another aspect of the invention regards the cDNA encoding for theconjugated cytokines herein disclosed, which can be prepared from thecytokines cDNA by addition of a 5′- or 3′-contiguous DNA sequenceencoding for the CD13 ligand, preferably for the homing peptidesdescribed above. The combined cDNA can be used as such or afterinsertion in vectors for gene therapy. The preparation and therapeuticapplications of suitable vectors is disclosed in (19), which is herebyincorporated by reference.

[0079] Polynucleotides for use in the invention comprise nucleic acidsequences encoding the polypeptide conjugate of the invention. It willbe understood by a skilled person that numerous differentpolynucleotides can encode the same polypeptide as a result of thedegeneracy of the genetic code. In addition, it is to be understood thatskilled persons may, using routine techniques, make nucleotidesubstitutions that do not affect the polypeptide sequence encoded by thepolynucleotides of the invention to reflect the codon usage of anyparticular host organism in which the polypeptides of the invention areto be expressed.

[0080] Polynucleotides of the invention may comprise DNA or RNA. Theymay be single-stranded or double-stranded. They may also bepolynucleotides which include within them synthetic or modifiednucleotides. A number of different types of modification tooligonucleotides are known in the art. These include methylphosphonateand phosphorothioate backbones, addition of acridine or polylysinechains at the 3′ and/or 5′ ends of the molecule. For the purposes of thepresent invention, it is to be understood that the polynucleotidesdescribed herein may be modified by any method available in the art.Such modifications may be carried out in order to enhance the in vivoactivity or life span of polynucleotides of the invention.

[0081] Polynucleotides of the invention can be incorporated into arecombinant replicable vector. The vector may be used to replicate thenucleic acid in a compatible host cell. Thus in a further embodiment,the invention provides a method of making polynucleotides of theinvention by introducing a polynucleotide of the invention into areplicable vector, introducing the vector into a compatible host cell,and growing the host cell under conditions which bring about replicationof the vector. The vector may be recovered from the host cell. Suitablehost cells include bacteria such as E. coli, yeast, mammalian cell linesand other eukaryotic cell lines, for example insect Sf9 cells.

[0082] Preferably, a polynucleotide of the invention in a vector isoperably linked to a control sequence that is capable of providing forthe expression of the coding sequence by the host cell, i.e. the vectoris an expression vector. The term “operably linked” means that thecomponents described are in a relationship permitting them to functionin their intended manner. A regulatory sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences.

[0083] The control sequences may be modified, for example by theaddition of further transcriptional regulatory elements to make thelevel of transcription directed by the control sequences more responsiveto transcriptional modulators.

[0084] Vectors of the invention may be transformed or transfected into asuitable host cell as described below to provide for expression of aprotein of the invention. This process may comprise culturing a hostcell transformed with an expression vector as described above underconditions to provide for expression by the vector of a coding sequenceencoding the protein, and optionally recovering the expressed protein.

[0085] The vectors may be for example, plasmid or virus vectors providedwith an origin of replication, optionally a promoter for the expressionof the said polynucleotide and optionally a regulator of the promoter.The vectors may contain one or more selectable marker genes, for examplean ampicillin resistance gene in the case of a bacterial plasmid or aneomycin resistance gene for a mammalian vector. Vectors may be used,for example, to transfect or transform a host cell.

[0086] Control sequences operably linked to sequences encoding theprotein of the invention include promoters/enhancers and otherexpression regulation signals. These control sequences may be selectedto be compatible with the host cell for which the expression vector isdesigned to be used in. The term “promoter” is well-known in the art andencompasses nucleic acid regions ranging in size and complexity fromminimal promoters to promoters including upstream elements andenhancers.

[0087] The promoter is typically selected from promoters which arefunctional in mammalian cells, although prokaryotic promoters andpromoters functional in other eukaryotic cells may be used. The promoteris typically derived from promoter sequences of viral or eukaryoticgenes. For example, it may be a promoter derived from the genome of acell in which expression is to occur. With respect to eukaryoticpromoters, they may be promoters that function in a ubiquitous manner(such as promoters of a-actin, b-actin, tubulin) or, alternatively, atissue-specific manner (such as promoters of the genes for pyruvatekinase). Tissue-specific promoters specific for certain cells may alsobe used. They may also be promoters that respond to specific stimuli,for example promoters that bind steroid hormone receptors. Viralpromoters may also be used, for example the Moloney murine leukaemiavirus long terminal repeat (MMLV LTR) promoter, the rous sarcoma virus(RSV) LTR promoter or the human cytomegalovirus (CMV) IE promoter.

[0088] It may also be advantageous for the promoters to be inducible sothat the levels of expression of the heterologous gene can be regulatedduring the life-time of the cell. Inducible means that the levels ofexpression obtained using the promoter can be regulated.

[0089] In addition, any of these promoters may be modified by theaddition of further regulatory sequences, for example enhancersequences. Chimeric promoters may also be used comprising sequenceelements from two or more different promoters described above.

[0090] Vectors and polynucleotides of the invention may be introducedinto host cells for the purpose of replicating thevectors/polynucleotides and/or expressing the proteins of the inventionencoded by the polynucleotides of the invention. Although the proteinsof the invention may be produced using prokaryotic cells as host cells,it is preferred to use eukaryotic cells, for example yeast, insect ormammalian cells, in particular mammalian cells.

[0091] Vectors/polynucleotides of the invention may introduced intosuitable host cells using a variety of techniques known in the art, suchas transfection, transformation and electroporation. Wherevectors/polynucleotides of the invention are to be administered toanimals, several techniques are known in the art, for example infectionwith recombinant viral vectors such as retroviruses, herpes simplexviruses and adenoviruses, direct injection of nucleic acids andbiolistic transformation.

[0092] Host cells comprising polynucleotides of the invention may beused to express conjugates of the invention. Host cells may be culturedunder suitable conditions which allow expression of the polypeptides andconjugates of the invention. Expression of the products of the inventionmay be constitutive such that they are continually produced, orinducible, requiring a stimulus to initiate expression. In the case ofinducible expression, protein production can be initiated when requiredby, for example, addition of an inducer substance to the culture medium,for example dexamethasone or IPTG.

[0093] Conjugates of the invention can be extracted from host cells by avariety of techniques known in the art, including enzymatic, chemicaland/or osmotic lysis and physical disruption.

DESCRIPTION OF THE FIGURES

[0094]FIG. 1: Effect of TNF and NGR-TNF on the growth of RMA-T lymphomas(a and b) and on the animal weight (c and d).

[0095] 5 Animals/group were treated with a single dose of TNF or NGR-TNF(i.p.), 10 days after tumor implantation. Tumor area values at day 14 asa function of the dose (b) and the loss of weight after treatment (meanof days 11 and 12) (d), were interpolated from logarithmic curves. Theanti-tumor effects induced by 1 μg or 9 μg of NGR-TNF at day 14 weregreater than those induced by comparable amounts of TNF (P=0.024 andP=0.032, respectively), while the loss of weight after these treatmentswas similar. The arrows indicate extrapolated doses of TNF and NGR-TNFthat induce comparable effects.

[0096]FIG. 2: Effect of mAb R3-63 and CNGRC on the anti-tumor activityof NGR-TNF (a) and TNF (b).

[0097] MAb R3-63 or CNGRC were mixed with NGR-TNF or TNF andadministered to RMA-T tumor bearing animals, 12 days after tumorimplantation (n=5 animals/group). In a separate experiment (c) TNF andNGR-TNF were coadministered with CNGRC or CARAC (a control peptide) toanimals bearing 11-day old tumors (n=5). The anti-tumor effect of 1 μgof NGR-TNF was stronger than that of 9 μg of TNF (P=0.009, t-test at day20) and was significantly inhibited by CNGRC (P=0.035) and by mAb R3-63(P=0.011).

[0098]FIG. 3: Effect of limited tryptic digestion of NGR-TNF and TNF ontheir mass (a) and anti-tumor activity (b).

[0099] Trypsin-agarose was prepared by coupling 1 mg of trypsin to 1 mlof Activated CH Sepharose (Pharmacia-Upjohn), according to themanufacturer's instructions. NGR-TNF and TNF (170 μg each in 300 μl of0.15 M sodium chloride, 0.05 M sodium phosphate, pH 7.3) were mixed with15 μl of resin suspension (1:4) or buffer alone and rotated end-over-endat 37° C. for the indicated time. The four products were filteredthrough a 0.22 μm Spin-X device (Costar, Cambridge, Mass.) and stored at−20° C. until use. (a) Electrospray mass spectrometry analysis. Themolecular mass values and the corresponding products (N-terminalsequences) are indicated on each peak. The arrows on the sequencesindicate the site of cleavage. (b) Effect of 1 or 3 μg of NGR-TNF andTNF, incubated without (upper panels) or with (lower panels) trypsin, onthe growth of RMA-T tumors and animal weight (mean±SE of groups treatedwith 1 and 3 μg doses). Animals were treated 13 days after tumorimplantation (n=5 animals/group).

[0100]FIG. 4: SDS-PAGE and anti-tumor activity of human NGR-TNF beforeand after denaturation/refolding.

[0101] SDS-PAGE under non reducing conditions (A) of human TNF (a),NGR-TNF before (b) and after (c) the denaturation/refolding processdescribed in Example V.

[0102] Effect of TNF and non-refolded NGR-TNF on the growth of RMA-Tlymphomas (B) and on body weight (C). Effect of human TNF (D) andrefolded NGR-TNF (consisting of >95% trimers with intra-chaindisulfides) (E) on the tumor growth. Animals (15 or 5 mice/group asindicated in each panel) were treated with one i.p. dose of TNF orNGR-TNF, 10 days after tumor implantation.

[0103]FIG. 5: Effect of a neutralising anti-mIFNγ antibody (AN18) on theantitumor activity of NGR-mTNF and doxorubicin on B16F1 tumors in C57BL6mice.

[0104]FIG. 6: Effect of NGR-TNF and doxorubicin in IFN knock out mice.

[0105]FIG. 7: Effect of NGR-mTNF, mIFNγ and doxorubicin (alon or incombinantion) on B16F1 tumors in nude mice.

[0106]FIG. 8: Effect of NGR-TNF and doxorubicin in nude mice

[0107]FIG. 9: Effect of NGR-mTNF, mIFNγ and doxorubicin on B16F1 tumorsin immunocompetent mice (C57BL6).

[0108]FIG. 10: IFN increase the penetration of doxorubicin in tumourswhen combined with NGR-TNF in IFN knock out animals.

The following examples further illustrate the invention. EXAMPLE IPreparation of Murine TNF and NGR-TNF

[0109] Murine recombinant TNF and Cys-Asn-Gly-Arg-Cys-Gly-TNF (NGR-TNF)were produced by cytoplasmic cDNA expression in E.coli. The cDNA codingfor murine Met-TNF₁₋₁₅₆ (20) was prepared by reversetranscriptase-polymerase chain reaction (RT-PCR) on mRNA isolated fromlipopolysaccharide-stimulated murine RAW-264.7 monocyte-macrophagecells, using 5′-CTGGATCCTCACAGAGCAATGACTCCAAAG-3′ and5′-TGCCTCACATATGCTCAGATCATCTTCTC-3′, as 3′ and 5′ primers.

[0110] The amplified fragment was digested with Nde I and Bam HI (NewEngland Biolabs, Beverley, Mass.) and cloned in pET-11b (Novagen,Madison, Wis. ), previously digested with the same enzymes (pTNF).

[0111] The cDNA coding for Cys-Asn-Gly-Arg-Cys-Gly-TNF₁₋₁₅₆ wasamplified by PCR on pTNF, using5′-GCAGATCATATGTGCAACGGCCGTTGCGGCCTCAGATCATCTTCTC-3′ as 5′ primer, andthe above 3′ primer. The amplified fragment was digested and cloned inpET-11b as described above and used to transform BL21(DE3) E.coli cells(Novagen). The expression of TNF and NGR-TNF was induced withisopropyl-β-D-thiogalactoside, according to the pET11b manufacturer'sinstruction. Soluble TNF and NGR-TNF were recovered from two-litercultures by bacterial sonication in 2 mM ethylenediaminetetracetic acid,20 mM Tris-HCl, pH 8.0, followed by centrifugation (15000×g, 20 min, 4°C.). Both extracts were mixed with ammonium sulfate (25% of saturation),left for 1 h at 4° C., and further centrifuged, as above. The ammoniumsulfate in the supernatants was then brought to 65% of saturation, leftat 4° C. for 24 h and further centrifuged. Each pellet was dissolved in200 ml of 1 M ammonium sulfate, 50 mM Tris-HCl, pH 8.0, and purified byhydrophobic interaction chromatography on Phenyl-Sepharose 6 Fast Flow(Pharmacia-Upjohn) (gradient elution, buffer A: 50 mM sodium phosphate,pH 8.0, containing 1 M ammonium sulfate; buffer B: 20% glycerol, 5%methanol, 50 mM sodium phosphate, pH 8.0). Fractions containing TNFimmunoreactive material (by western blotting) were pooled, dialyzedagainst 2 mM ethylenediaminetetracetic acid, 20 mM Tris-HCl, pH 8.0 andfurther purified by ion exchange chromatography on DEAE-Sepharose FastFlow (Pharmacia-Upjohn) (gradient elution, buffer A: 20 mM Tris-HCl, pH8.0; buffer B: 1 M sodium chloride, 20 mM Tris-HCl, pH 8.0). Fractionscontaining TNF-immunoreactivity were pooled and purified by gelfiltration chromatography on Sephacryl-S-300 HR (Pharmacia-Upjohn),pre-equilibrated and eluted with 150 mM sodium chloride, 50 mM sodiumphosphate buffer, pH 7.3 (PBS). Fractions corresponding to 40000-50000Mr products were pooled, aliquoted and stored frozen at −20° C. Allsolutions employed in the chromatographic steps were prepared withsterile and endotoxin-free water (Salf, Bergamo, Italy). The finalyields were 45 mg of TNF and 34.5 mg NGR-TNF.

[0112] The molecular weight of purified TNF and NGR-TNF was measured byelectrospray mass spectrometry. The protein content was measured using acommercial protein assay kit (Pierce, Rockford, Ill.). Endotoxin contentof NGR-TNF and TNF was 0.75 units/μg and 1.38 units/μg, respectively, asmeasured by the quantitative chromogenic Lymulus Amoebocyte Lysate (LAL)test (BioWhittaker).

[0113] Sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and western blot analysis were carried out using 12.5 or 15%polyacrylamide gels, by standard procedures.

[0114] A small amount of TNF and NGR-NF was further purified by affinitychromatography on soluble p55-TNF receptor (sTNF-R1)-Sepharose asfollows: 5 mg of recombinant sTNF-R1 were prepared as described (22) andcoupled to 2 ml of Activated-CH-Sepharose (Pharmacia), according to themanufacturer's instruction. Two separate columns (one ml each), werewashed extensively with sterile and endotoxin-free solutions, loadedwith purified TNF or NGR-TNF in PBS and desorbed by gradient elution (1h, buffer A: PBS; buffer B: 0.5 M sodium chloride, 0.2 M glycine-HCl).The TNF-antigen containing fractions were neutralized and dialyzedagainst sterile physiological solution. Endotoxin-free human serumalbumin was added before dialysis (0.5 mg/ml) to prevent proteinadsorption on membranes. The TNF content in each fraction was measuredby ELISA and cytolytic assay.

[0115] Non reducing SDS-PAGE of TNF showed a single band of 17-18 kDa,as expected for monomeric TNF (not shown). At variance, non reducingSDS-PAGE and western blot analysis of NGR-TNF showed differentimmunoreactive forms of 18, 36 and 50 kDa likely corresponding tomonomers, dimers and trimers. Under reducing conditions most of the 50and 36 kDa bands were converted into the 18 kDa form, pointing to thepresence of NGR-TNF molecules with interchain disulfide bridges. The 18kDa band accounted to about ⅔ of the total material, whereas the 36 kDaaccounted for most of the remaining part. These electrophoretic patternssuggest that NGR-TNF was a mixture of trimers made up by three monomericsubunits with correct intra-chain disulfides (at least 50%) and theremaining part mostly by trimers with one or more interchain disulfides.The 36 kDa band still observed by reducing SDS-PAGE suggests thatNGR-TNF contained also an irreversible denatured dimer (about 10% oftotal).

[0116] The molecular mass of TNF and NGR-TNF monomers were 17386.1±2.0Da and 17843.7±2.5 Da, respectively, by electrospray mass spectrometry.These values correspond very well to the mass expected for Met-TNF₁₋₁₅₆(17386.7 Da) and for CNGRCG-TNF₁₋₁₅₆ (17844.2 Da).

EXAMPLE II In Vitro Cytotoxic Activity of Murine TNF and NGR-TNF

[0117] The bioactivity of TNF and NGR-TNF was estimated by standardcytolytic assay based on L-M mouse fibroblasts (ATCC CCL1.2) asdescribed (23). The cytolytic activity of TNF and NGR-TNF on RMA-T cellswas tested in the presence of 30 ng/ml actinomycin D. Each sample wasanalyzed in duplicate, at three different dilutions. The results areexpressed as mean±SD of two-three independent assays.

[0118] The in vitro cytotoxic activity of TNF and NGR-TNF was(1.2±0.14)×10⁸ units/mg and (1.8±0.7)×10⁸ units/mg, respectively, bystandard cytolytic assay with L-M cells. These results indicate that theCNGRCG moieties in the NGR-TNF molecule does not prevent folding,oligomerization and binding to TNF receptors.

[0119] In a previous study we showed that RMA-T cells can be killed byTNF in the presence of 30 ng/ml actinomycin D, whereas in the absence oftranscription inhibitors these cells are resistant to TNF, even afterseveral days of incubation. The in vitro cytotoxic activity of NGR-TNFon RMA-T cells in the presence of actinomycin D was (1.4±0.8)×10⁸units/mg, as measured using TNF ((1.2±0.14)×10⁸ units/mg) as a standard.Thus, the cytotoxic activities of NGR-TNF and TNF were similar both onL-M and RMA-T cells.

EXAMPLE III Characterization of the Therapeutic and Toxic Activity ofMurine TNF and NGR-TNF

[0120] The Rauscher virus-induced RMA lymphoma of C57BL/6 origin, weremaintained in vitro in RPMI 1640, 5% foetal bovine serum (FBS), 100 U/mlpenicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B, 2 mMglutamine and 50 μM 2-mercaptoethanol. RMA-T was derived from the RMAcell line by transfection with a construct encoding the Thy 1.1 alleleand cultured as described (14).

[0121] B16F1 melanoma cells were cultured in RPMI 1640, 5% FBS, 100 U/mlpenicillin, 100 μml streptomycin, 0.25 μg/ml amphotericin B, 2 mMglutamine, 1% MEM non essential amino acid (BioWhittaker Europe,Verviers, Belgium).

[0122] In vivo studies on animal models were approved by the EthicalCommittee of the San Raffaele H Scientific Institute and performedaccording to the prescribed guidelines. C57BL/6 (Charles RiverLaboratories, Calco, Italy) (16-18 g) were challenged with 5×10⁴ RMA-Tor B16F1 living cells, respectively, s.c. in the left flank. Ten-twelvedays after tumor implantation, mice were treated, i.p., with 250 μl TNFor NGR-TNF solutions, diluted with endotoxin-free 0.9% sodium chloride.Preliminary experiments showed that the anti-tumor activity was notchanged by the addition of human serum albumin to TNF and NGR-TNFsolutions, as a carrier. Each experiment was carried out with 5 mice pergroup. The tumor growth was monitored daily by measuring the tumor sizewith calipers. The tumor area was estimated by calculating r₁×r₂,whereas tumor volume was estimated by calculating r₁×r₂×r₃×{fraction(4/3)}, where r₁ and r₂ are the longitudinal and lateral radii, and r₃is the thickness of tumors protruding from the surface of normal skin.Animals were killed before the tumor reached 1.0-1.3 cm diameter. Tumorsizes are shown as mean±SE (5-10 animals per group as indicated in thefigure legends) and compared by t-test.

[0123] The anti-tumor activity and toxicity of NGR-TNF were compared tothose of TNF using the RMA-T lymphoma and the B16F1 melanoma models inC57BL6 mice. Since the RMA-T model has been previously characterized andused to study the anti-tumor activity of TNF with different targetingprotocols (26) we decided to use this model also in this study.

[0124] Murine TNF administered to animals bearing established s.c. RMA-Ttumors, causes 24 h later a reduction in the tumor mass and haemorragicnecrosis in the central part of the tumor, followed by a significantgrowth delay for few days (26). A single treatment with TNF does notinduce complete regression of this tumor, even at doses close to theLD50, as living cells remaining around the necrotic area restart to growfew days after treatment.

[0125] In a first set of experiments we investigated the effect ofvarious doses (i.p.) of TNF or NGR-TNF on animal survival. To avoidexcessive suffering, the animals were killed when the tumor diameter wasgreater than 1-1.3 cm. The lethality of TNF and NGR-TNF, 3 days aftertreatment, was similar (LD50, 60 μg and 45 μg, respectively) whereastheir anti-tumor activity was markedly different (Table 1). TABLE 1Survival of mice with RMA-T lymphoma treated with murine TNF or NGR-TNFSurvival (%)^(a)) after treatment Day Day Animals Dose Day Day Day DayDay 38 62 Day Treatment (n) (μg) 3 7 14 21 30 (2^(nd) ch)^(b)) (3°ch.)^(b)) 92 None 18 0 100 0 TNF 4 1 100 20 0 9 3 100 55 0 9 9 100 55 2211 0 14 27 100 57 14 7 0 9 54 55 55 0 9 108 0 NGR-TNF 10 1 100 70 10 1010 0 10 3 100 80 20 20 20 0 9 9 100 88 55 22 11 11 11 13 27 100 85 30 2315 15 15 11 9 54 33 33 0 15 9 108 0

[0126] For instance, 1 or 3 μg of NGR-TNF delayed tumor growth moreefficiently then 27 μg of TNF, indicating that NGR-TNF was at least oneorder of magnitude more active. Interestingly, some animals were curedwith doses of NGR-TNF lower than the LD50, whereas no animals at allwere cured with TNF. Cured animals rejected further challenges withtumorigenic doses of either RMA-T or wild-type RMA cells, suggestingthat a single treatment with NGR-TNF was able to induce protectiveimmunity. It is noteworthy that increasing the dose of TNF or NGR-TNFabove 9-27 μg markedly increased the toxicity and poorly or not thetherapeutic activity.

[0127] The loss of weight consequent to TNF treatment is a well knownsign of systemic toxicity (26 ). Thus, to further compare theefficacy/toxicity ratio of TNF and NGR-TNF we monitored the tumor growthand the animal weight after treatment. The effect of 1 μg of NGR-TNF onthe tumor growth was similar or higher than that of 9 μg of TNF (FIG.1a), while the loss of weight one-two days after treatment wascomparable to that of 1 μg of TNF (FIG. 1c). When we interpolated thedata with a logarithmic curve in a dose-response plot we found that thetherapeutic effect of 9 μg of TNF at day 14 can be obtained with aslittle as 0.6 μg of NGR-TNF (FIG. 1b). In contrast, 8.5 μg werenecessary to induce a comparable toxic effect (FIG. 1d). Thus, thecalculated efficacy/toxicity ratio of NGR-TNF under these conditions is14 times greater than that of TNF.

[0128] Similar results were obtained with the B16F1 melanoma model.Treatment with 1 μg of NGR—TNF at day 11 and day 17, induced ananti-tumor response at day 19 greater than that obtained with 4 μg ofTNF and similar to that obtained with 12 μg of TNF (data not shown). Incontrast, the loss of weight caused by 1 μg of NGR-TNF was markedlylower than that caused by 4 and 12 μg of TNF. Treatment with 12 μg ofNGR-TNF caused an even stronger anti-tumor effect, while the toxiceffect was similar to that of 12 μg of TNF.

[0129] When a third injection was done on day 19 some animal deathsoccurred 1-2 days later in all groups (2 out of 5 in the group treatedwith saline and 12 μg of NGR-TNF and 1 out of 5 in the remaininggroups). Of note, one animal treated with 12 μg of NGR-TNF completelyrejected the tumor. When this animal was challenged with a secondtumorigenic dose of B16F1 cells, a palpable tumor developed after 18days, while control animals developed a tumor within 6-7 days.

[0130] These results, altogether, suggest that the efficacy of NGR-TNFin inhibiting the tumor growth is 10-15 times greater than that of TNFwhereas the toxicity is similar. Moreover, NGR-TNF can induce protectiveimmune responses more efficiently than TNF.

EXAMPLE IV Mechanism of Action of NGR-TNF

[0131] Anti-mouse CD13 mAb R3-63 purified from ascitic fluids byprotein-G Sepharose chromatography (Pharmacia-Upjohn, Uppsala, Sweden),and dialyzed against 0.9% sodium chloride.

[0132] Rabbit polyclonal antiserum was purchased from Primm srl (Milan,Italy) and purified by affinity chromatography on protein-A-Sepharose(Pharmacia-Upjohn). CNGRC and CARAC peptides were prepared as describedpreviously (28).

[0133] To provide evidence that the improved activity of NGR-TNF isdependent on tumor targeting via the NGR moiety we have investigatedwhether the in vivo activity of NGR-TNF can be partially competed byCNGRC. To this end we have administered NGR-TNF (1 μg) to RMA-T tumorbearing mice, with or without a molar excess of CNGRC. In parallel,other animals were treated with TNF (3 or 9 μg), again with or withoutCNGRC. As expected, CNGRC decreased significantly the anti-tumoractivity of NGR-TNF (FIG. 2a) but not that of TNF (FIG. 2b). Atvariance, a control peptide (CARAC) was unable to cause significantdecrease of NGR-TNF activity (FIG. 2c). These results suggest that CNGRCcompetes for the binding of NGR-TNF to a CNGRC receptor, and support thehypothesis of a targeting mechanism for the improved activity. Of note,CNGRC was unable to decrease the in vitro cytotoxic activity of NGR-TNF(data not shown).

[0134] Since it has been recently reported that aminopeptidase N (CD13)is a receptor for CNGRC peptides, we then investigated the contribute ofthis receptor in the targeting mechanism of NGR-TNF. To this end, westudied the effect of an anti-CD13 mAb (R3-63) on the anti-tumoractivity of NGR-TNF and TNF. MAb R3-63 significantly inhibited theanti-tumor activity of NGR-TNF (FIG. 2a) but not that of TNF (FIG. 2b)indicating that CD13 is indeed critically involved in the anti-tumoractivity of NGR-TNF. No expression of CD13 on RMA-T cell surface wasobserved by FACS analysis of cultured cells with mAb R3-63 (not shown),suggesting that other cells were recognized by the antibody in vivo.

[0135] Although these data indicate that CD13 is an important receptorfor NGR-TNF, we cannot entirely exclude that binding to other not yetidentified NGR receptors also contribute, albeit to a lower extent, tothe targeting mechanism.

[0136] Preliminary experiments of partial proteolysis showed that theArg-Ser bond in the N-terminal segment of TNF (residues 2-3) is verysensitive to trypsin, whereas the rest of the molecule is much moreresistant. Thus, to provide further evidence that the improved activityof NGR-TNF is related to its NGR moiety, we tried to cleave out the NGRdomain from the N-terminal region of the mutein by partial digestionwith immobilized trypsin. This treatment converted both NGR-TNF and TNFinto a molecule corresponding to the TNF3-156 fragment (expected mass16986.2 Da; see FIG. 3a for measured mass and expected sequences).

[0137] While digestion did not decrease the in vitro cytolytic activityof NGR-TNF on L-M cells (2.3±1.4)33 10⁸ U/mg) its in vivo anti-tumoractivity was decreased to the level of TNF (FIG. 3b). Of note, thetoxicity of NGR-TNF and TNF were similar both before and afterdigestion, as judged from animal weight loss one day after treatment(FIG. 3b, right panel), suggesting that the NGR-dependent targetingmechanism does not alters the toxicity.

EXAMPLE V Preparation and Characterization of Human TNF and NGR-TNF

[0138] Human recombinant TNF and NGR-TNF (consisting of human TNF1-157fused with the C terminus of CNGRCG) were prepared by recombinant DNAtechnology and purified essentially as described for murine TNF andNGR-TNF. The cDNA coding for human NGR-TNF was prepared by PCR onplasmid pET11b/hTNF containing the hTNF coding sequence (33), using thefollowing primers:

[0139] NGR-hTNF/1 (sense): 5′A TAT CAT ATG TGC AAC GGC CGT TGC GGC GTCAGA TCA TCdT TCT CG 3′.

[0140] NGR-hTNF/2 (antisense): 5′ TCA GGA TCC TCA CAG GGC AAT GAT CCCAAA GTA GAC 3′.

[0141] The amplified fragment was digested and cloned in pET-11b (NdeI/BamH I) and used to transform BL21(DE3) E.coli cells (Novagen). Theexpression of NGR-hTNF was induced with isopropyl-β-D-thiogalactoside,according to the pET11b manufacturer's instruction. Soluble NGR-TNF wasrecovered from two-liter cultures by bacterial sonication in 2 mMethylenediaminetetracetic acid, 20 mM Tris-HCl, pH 8.0, followed bycentrifugation (15000×g, 20 min, 4° C.).

[0142] The extract was mixed with ammonium sulfate (35% of saturation),left for 1 h at 4° C., and further centrifuged, as above. The ammoniumsulfate in the supernatants was then brought to 65% of saturation, leftat 4° C. for 24 h and further centrifuged. Each pellet was dissolved in1 M ammonium sulfate, 50 mM Tris-HCl, pH 8.0, and purified byhydrophobic interaction chromatography on Phenyl-Sepharose 6 Fast Flow(Pharmacia-Upjohn) (gradient elution, buffer A: 100 mM sodium phosphate,pH 8.0, containing 1 M ammonium sulfate; buffer B: 70% ethylen glycol,5% methanol, 100 mM sodium phosphate, pH 8.0). Fractions containing hTNFimmunoreactive material (by ELISA) were pooled, dialyzed against 20 mMTris-HCl , pH 8.0 and further purified by ion exchange chromatography onDEAE-Sepharose Fast Flow (Pharmacia-Upjohn) (gradient elution, buffer A:20 mM Tris-HCl, pH 8.0; buffer B: 1 M sodium chloride, 20 mM Tris-HCl,pH 8.0). All solutions employed in the chromatographic steps wereprepared with sterile and endotoxin-free water (Salf, Bergamo, Italy).

[0143] At this point about 30 mg of TNF and 32 mg NGR-TNF was recoveredfrom two-liters cultures. Non reducing SDS-PAGE showed bandscorresponding to monomers, dimers and trimers suggesting that also humanNGR-TNF was a mixture of trimers with correct intra-chain disulfides andtrimers with one or more interchain disulfide bridges (FIG. 4A, lane b),as observed with murine NGR-TNF.

[0144] Trimers with correct intrachain disulfide bridges were isolatedfrom this mixture by a four-step denaturation-refolding process asfollows: purified human NGR-TNF was denatured with 7 M urea andgelfiltered through an HR Sephacryl S-300 column (1025 ml) (Pharmacia)pre-equilibrated with 7 M urea, 100 mM Tris-HCl, pH 8.0. Fractionscorresponding to monomeric TNF were pooled, ultrafiltered through an YMMWCO 10 kDa membrane (Amicon) and refolded by dialysis against 33volumes of 2.33 M urea, 100 mM Tris-HCl, pH 8 at 4° C. (140 min)followed by 1.55 M urea, 100 mM Tris-HCl, pH 8 (140 min) and 1 M urea,100 mM Tris-HCl, pH 8 (140 min). Finally the product was dialyzedagainst 80 volumes of 100 mM Tris-HCl (16 h), centrifuged at 13000×g (30min), filtered through a SFCA 0.45 μm membrane (Nalgene) and gelfilteredthrough an HR Sephacryl S-300 column (1020 ml) pre-equilibrated with0.15 M sodium chloride, 0.05 M sodium phosphate (PBS). About 23 mg ofrefolded protein was recovered.

[0145] The final product was mostly monomeric after non reducingSDS-PAGE (FIG. 4A, lane c), had an hydrodynamic volume similar to thatof trimeric human TNF by analytical gel-filtration HPLC on a Superdex 75HR column (not shown), and had a molecular mass of 17937.8+1.8 Da(expected for CNGRCG-TNF1-157, 17939.4 Da) by electrospray massspectrometry. The in vitro cytolytic activities of non-refolded andrefolded NGR-TNF on mouse L-M cells were (6.11×107)+4.9 and(5.09×107)+0.3 units/mg respectively, whereas that of purified human TNFwas (5.45×107)+3.1 units/mg. These results suggest that thedenaturation-refolding process did not affect the interaction of humanNGR-TNF with the murine p55 receptor.

[0146] The in vivo anti-tumor activity of 1 μg of human NGR-TNF (nonrefolded) was greater than that of 10 μg of TNF (FIG. 4B) whereas thetoxicity, as judged by animal weight loss, was significantly lower (FIG.4C). After refolding 0.3 μg of NGR-TNF was sufficient to induce ananti-tumor effect stronger than that achieved with 10 μg of TNF (FIGS.4D, 4E).

[0147] These results indicate that the anti-tumor activity of humanNGR-TNF is greater than that of human TNF.

[0148] Furthermore, we have observed that refolded human and mouseNGR-TNF can induce significant anti-tumor effects on RMA-T-bearing miceeven at very low doses (1-10 ng/mouse) with no evidence of toxiceffects, while TNF was unable to induce significant effects at thesedoses (not shown).

EXAMPLE VI Preparation and Characterization of Mouse NGR-IFNγ

[0149] Recombinant murine interferon (IFN)γ fused with CNGRCG (NGR-IFNγ)was prepared by recombinant DNA technology, essentially as described forNGR-TNF. The CNGRC domain was fused with the C terminus of IFNγ.Moreover the cysteine in position 134 was replaced with a serine; amethionine was introduced in position −1 for expression in E.coli cells.The PCR primers used for the production of the NGR-IFNγ cDNA were: 5′-ATAT CTA CAT ATG CAC GGC ACA GTC ATT GAA AGC C (sense) and 5′-TC GGA TCCTCA GCA ACG GCC GTT GCA GCC GGA GCG ACT CCT TTT CCG CTT CCT GAG GC. ThecDNA was cloned cloned in pET-11b (Nde I/BamH I) and used to transformBL21(DE3) E.coli cells (Novagen). Protein expression was induced withisopropyl-β-D-thiogalactoside, according to the pET11b manufacturer'sinstruction. The product was purified from E.coli extracts byimmunoaffinity chromatography using an anti-mouse IFNγ mAb (AN18)immobilized on agarose, according to standard techniques. Reducing andnon reducing SDS-PAGE of the final product showed a single band of 16kDa. Electrospray mass spectrometry showed a molecular weight of16223+3.6 Da (expected, 1625.5 Da) corresponding to murineMet-IFNγ1-134(C134S)CNGRC (NGR-IFNγ).

[0150] The capability of NGR-IFNγ and NGR-TNF to compete the binding ofan anti-CD13 antibody to tumor associated vessels was investigated byusing an immunohistochemical approach.

[0151] Fresh surgical specimens of human renal cell carcinoma wereobtained from the Histopathology Department of the San Raffaele HScientific Institute. Sections (5-6 μm thick) of Bouin-fixed (4-6 h)paraffin-embedded specimens were prepared and adsorbed onpolylysine-coated slides. CD13 antigen were detected using theavidin-biotin complex method as follows: tissue sections were rehydratedusing xylenes and graded alcohol series, according to standardprocedures. Tissue sections were placed in a vessel containing 1 mM EDTAand boiled for 7 min using a micro-wave oven (1000 W). The vessel wasthen refilled with 1 mM EDTA and boiled again for 5 min. The tissuesections were left to cool and incubated in PBS containing 0.3% hydrogenperoxide for 15 min, to quench endogenous peroxidase. The samples werethen and rinsed with PBS and incubated with 100-200 μl of PBS-BSA (1 hat room temperature) followed by the mAb WM15 (anti-hCD13), alone ormixed with various competitor agents (see Table 2) in PBS-BSA (overnightat 4° C.). The slides were then washed 3 times (3 min each) with PBS andincubated with PBS-BSA containing 2% normal horse serum (PBS-BSA-NHS)(Vector Laboratories, Burlingame, Calif.) for 5 min. The solution wasthen replaced with 3 μg/ml biotinylated horse anti-mouse IgG (H+L)(Vector Laboratories, Burlingame, Calif.) in PBS-BSA-NHS and furtherincubated for 1 h at room temperature. The slides were washed again andincubated for 30 min with Vectastain Elite Reagent (Vector Laboratories,Burlingame, Calif.) diluted 1:100 in PBS. A tablet of3,3′-diamino-benzidine-tetrahydrocloride (Merck, Darmstadt, Germany) wasthen dissolved in 10 ml of deionized water containing 0.03% hydrogenperoxide, filtered through a 0.2 μm membrane and overlaid on tissuesections for 5-10 min. The slides were washed as above andcounterstained with Harris' hematoxylin. The tumor associated vesselswere identified by staining serial sections of the tissue with ananti-CD31 mAb (mAb JC/70A, anti-human CD31, IgG1, from DAKO, Copenhagen,Denmark).

[0152] The results are summarized in Table 2. As shown, the binding ofWM15 to tumor associated vessels was inhibited by an excess of NGR-TNF,NGR-IFNγ and CNGRC, but not by other control reagents lacking the NGRmotif. This suggests that the NGR binding site on CD13 stericallyoverlaps with the WM15 epitope. In contrast, NGR-TNF was unable tocompete the binding of 13C03 to epithelial cells.

[0153] We conclude that the NGR moiety of NGR-IFNγ and NGR-TNF and caninteract with a CD13 form recognized by mAb WM15 on tumor associatedvessels. Moreover, these results indicate that the CNGRC motif isfunctional either when linked to the N-terminus or the C-terminus of acytokine. TABLE 2 Binding of WM15 to renal cell cancer sections in thepresence of various competitors Binding of WM15 to tumor Competitorassociated vessels None + NGR-TNF (25 μg/ml) − NGR-IFNγ (50 μg/ml) −CNGRC (100 μg/ml) − TNF (25 μg/ml) + Human serum albumin (25 μg/ml) +Synthetic CgA(60-68) (100 μg/ml) +

EXAMPLE VII Targeted Delivery of Biotinylated NGR-TNF to Tumors UsingAnti-Tumor Antibodies and Avidin (Pre-Targeting)

[0154] The following example illustrates the possibility of “dual”targeting of TNF, based on the combination of a tumor homing antibodyand the peptide CNGRC.

[0155] A biotin-NGR-TNF conjugate was prepared by mixing NGRTNF withD-biotinyl-6-aminocaproic acid N-hydroxysuccinimide ester (SocietáProdotti Antibiotici S.p.A, Milan, Italy), in 1 M sodium-carbonatebuffer, pH 6.8 (3 h at room temperature) (21). The reaction was blockedwith 1 M Tris-HCl, pH 7.5.

[0156] The conjugate was characterized by mass spectrometry and found tocontain 1 biotin/trimer (on average). C57BL/6 (Charles RiverLaboratories, Calco, Italy) were then challenged with 5×10⁴ RMA-T livingcells, s.c. in the left flank. When the tumor area reached 40 mm², micewere treated by sequential injections of biotinylated antibody, avidinsand biotin-TNF according to a “three-day” protocol as describedpreviously (26). We injected: 40 μg biotin-mAb19E12 (i.p., step I), 60μg avidin and 60 μg streptavidin after 18 and 19 h, respectively (i.p.,step II), 3 μg of biotin-NGR-TNF, 24 h later (i.p, step III). Eachcompound was diluted with a sterile 0.9% sodium chloride solution. Incontrol experiment, avidin and streptavidin were omitted. Eachexperiment was carried out with 5 mice/group. The tumor growth wasmonitored daily by measuring the tumor size with calipers. The tumorareas before and 10 days after treatment were 39±4 mm² and 8±5 mm²,respectively, in the group treated with mAb19E12-biotin/avidin/streptavidin/biotin-NGR-TNF (5 animals, mean±SE) .In the control group (treated with mAb 19E12-biotin /biotin-NGR-TNFalone) the tumor areas before and 10 days after treatment were 40±4 mm²and 20±6 mm² respectively, indicating that pre-targeting with tumorhoming antibody and avidin has increased the activity of NGR-TNF.

EXAMPLE VIII Synergistic Activity Between NGR-TNF and Interferon-γ

[0157] Mouse B16F1 melanoma cells were cultured as described previously(Curnis et al., 2000; Moro et al., 1997).

[0158] The neutralizing anti-IFNgamma monoclonal antibody AN18 waskindly supplied by P. Dellabona, Milan, Italy). Doxorubicin(Adriblastina) was purchased from Pharmacia-Upjohn (Milan, Italy).Recombinant murine IFNγ was purchased from Peprotech Inc. (USA)(endotoxin content: <1 Units/μg). TS/A cells from a BALB/c spontaneousmammary adenocarcinoma were cultured in RPMI 1640 medium, 10% fetalbovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/mlamphotericin B, 2 mM glutamine and 1% minimal essential medium withnonessential amino acids (BioWhittaker Europe, Verviers, Belgium).

[0159] Murine TNF and NGR-TNF (consisting of TNF fused with theC-terminus of CNGRCG) was prepared by recombinant DNA technology andpurified from E.coli cell extracts, as described (Curnis, et al., 2000).All solutions used in the chromatographic steps were prepared withsterile and endotoxin-free water (Salf, Bergamo, Italy). Proteinconcentration was measured with a commercial protein quantificationassay kit (Pierce, Rockford, Ill.). The cytolytic activity of NGR-mTNFwas 9.1×10⁷ Units/mg. The hydrodynamic volume of NGR-mTNF was similar tothat of mTNF, a homotrimeric protein (Smith and Baglioni, 1987), by gelfiltration chromatography on a Superdex 75 HR column (Pharmacia,Sweden). Endotoxin content of NGR-mTNF was 0.082 Units/μg.

[0160] DNA manipulations were performed by standard recombinant DNAmethods. The cDNA coding for murine IFNγ was prepared by PCR on cDNAobtained from murine lymphocytes stimulated with phorbol 12-myristate13-acetate, using the following primers: 5′AGAATTCATGAACGCTACACACTGCATCTTGGC 3′ (forward primer); 5′TATATTAAGCTTTCAGCAGCGACTCCTTTTCCGC 3′ (reverse primer). Primers weredesigned to amplify the cDNA sequence coding for the full-length mouseIFNγ, including the leader sequence. They include the EcoRI and HindIIIrestriction site (underlined) for the cloning into the mammalianexpression vector pRS 1-neo, to generate pRS1 neo-IFNγ. pRS1 neo-IFNγwas then prepared using the Plasmid Maxi Kit (Qiagen Inc.-Diagen, GmbH,Germany) and diluted at 1 mg/ml in sterile and endotoxin-free water(S.A.L.F. Laboratorio Farmacologico SpA, Bergamo, Italy). pRS1neo-IFNγ(3 μg) was mixed with 100 μl of 0.03 mg/ml Lipofectin Reagent (GibcoBrl) in RPMI 1640 and incubated for 20 min at room temperature. Then themixture was added to TS/A cells plated in 24-well microtiter plates 1day before (4×10⁴ cells per well in 200 μl of culture medium). Afterincubation at 37° C., 5% CO₂ for 4 h, 2 ml of culture medium were addedto each well. After 48 h of incubation the culture medium was changedwith RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamineand containing 1 mg/ml geneticin. One week later, cells survivingselection were cloned by limiting dilution in 96-well microtiter platesin the presence of geneticin. The supernatants of each clone was testedby IFNγ-ELISA. Ten IFNγ-secreting clones were obtained. One clone, namedTS/A-IFNγ, producing 1.13 μg IFNγ per ml of culture medium was selectedand used for in vivo experiments. IFNγ-ELISA. PVC microtiter plates(Becton Dickinson, cod.3912) were coated with 5 μg/ml mAb AN18 in PBS(16 h at 4° C.). Plates were washed three times with PBS and blockedwith 2% bovine serum albumin (BSA) in PBS (PBS-BSA, 1 h at 37° C.).Plates were then washed three times with PBS and incubated with cellculture supernatants or mouse IFNγ standard solutions diluted inPBS-BSA. Plates were washed eight times with PBS containing 0.05%Tween-20 (Merck) (PBS-TW) and incubated with mAb XMG1.2-bio (0.2 μg/mlin PBS, 1 h at 37° C.). Plates were washed again with PBS-TW andincubated for 1 h at 37° C. with streptavidin-horseradish peroxidase(Sigma) diluted 1:3000 in PBS-BSA. After washing with PBS-TW, boundperoxidase was detected with o-phenylenediamine dihydrochlorideperoxidase substrate (Sigma). The chromogenic reaction was blocked after30 min by adding 10% sulfuric acid. The A₄₉₀ was measured with an ELISAmicroplate reader (Biorad).

[0161] Studies on animal models were approved by the Ethical Committeeof the San Raffaele H Scientific Institute and performed according tothe prescribed guidelines. C57BL/6 mice (Charles River Laboratories,Calco, Italy) weighing 16-18 g were challenged with subcutaneousinjection in the left flank of 5×10⁴ or B16F1 living cells; 5 dayslater, the mice were treated with NGR-mTNF and mIFNγ solutions (100 μl)followed 2 h later by administration of doxorubicin solution (100 μl).All drugs were administered intraperitoneally (i.p). The drugs werediluted with 0.9% sodium chloride, containing 100 μg/ml endotoxin-freehuman serum albumin (Farma-Biagini, Lucca, Italy), except fordoxorubicin, which was diluted with 0.9% sodium chloride alone. Tumorgrowth was monitored daily by measuring the tumors with calipers aspreviously described (Gasparri et al., 1999). Animals were sacrificedbefore the tumors reached 1.0-1.5 cm in diameter. Tumor sizes are shownas mean±SE (5 animals/group).

[0162] Endogenous IFN is Critical for NGR-TNF/Doxorubicin TherapeuticActivity.

[0163] We have shown previously that NGR-mTNF and doxorubicin playsynergistic effects in B16-F1 tumor-bearing immunocompetent mice, theanti-tumor activity of doxorubinin being significantly increased bypre-administration of 0.1 ng of NGR-mTNF (above and Cumis et al., 2002).Accordingly, administration of low amounts of NGR-mTNF (0.1 ng) incombination with doxorubicin (80 μg) to B16-F10 tumor-bearing miceinduced anti-tumor effects stronger than those obtained with doxorubicinalone (FIG. 5). These drugs act synergistically, as NGR-TNF alone ispoorly active when used at low dosage (0.1 ng) (Curnis et al., 2002).

[0164] To assess the functional importance of endogenous mIFNγ in theanti-tumor activity of NGR-mTNF in immunocompetent mice, we studied theeffect of a neutralizing anti-mIFNγ antibody (mAb AN18) on theanti-tumor activity of NGR-mTNF/doxorubicin in C57BL6 mice.

[0165] When this antibody was administered 24 h before NGR-mTNF, thesynergistic effect between NGR-mTNF and doxorubicin was abolished (FIG.5), supporting the hypothesis that endogenous mIFNγ is an importantplayer in the anti-tumor activity of NGR-mTNF.

[0166] To further support the role of IFN in the therapeutic response tothese drugs we performed other in vivo experiments using BALB/cIFNγ^(−/−) “knock out” mice, bearing subcutaneous mouse TS/A-mammaryadenocarcinoma tumors. In parallel, we performed similar experimentsusing wild-type BALB/c IFNγ^(+/+) mice. NGR-TNF/doxorubicinsignificantly reduced the tumor mass in BALB/c IFNγ^(+/+) mice (FIG. 6,black bars), but not in BALB/c IFNγ^(−/−) mice (white bars). Notably,administration of exogenous IFN (300 ng) in combination with NGR-TNF anddoxorubicin to BALB/c IFNγ^(−/−) mice restored the synergy between thesedrugs (FIG. 6, white bars). These results confirm the hypothesis thatIFN is necessary for the NGR-TNF/doxorubicin synergistic activity.Co-administration of IFN and doxorubicin without NGR-TNF did not inducesignificant anti-tumor effects in BALB/c IFNγ^(−/−) mice (FIG. 6, whitebars) indicating that this cytokine acts synergistically with NGR-TNF,and little or not with doxorubicin.

[0167] Role of T-cells and Locally Produced IFN in NGR-TNF/DoxorubicinTherapeutic Activity.

[0168] T- and NK-cells are the primary sources of IFN in immunocompetentmice. To investigate the importance of T-cells as a source of the IFN inthe NGR-TNF/doxorubicin combined therapy we investigated the effect ofthese drugs in B16F 1 tumor-bearing nu/nu mice, lacking T-cells. In thismodel the NGR-TNF/doxorubicin synergistic activity was lost (FIGS.7A-C). However, when these drugs were administered in combination withIFN, the synergistic effect was observed again (FIG. 7D). It is likelythat the amount of endogenous IFN in these animals was not sufficient toactivate the NGR-TNF/doxorubicin synergism, while administration ofexogenous IFN restored the synergy.

[0169] The results of in vivo experiments with immunocompetent and nu/numice may suggest that T-cells present within the tumor mass ofimmunocompetent mice, but not of nu/nu mice, produce sufficient amountsof IFN to stimulate a synergistic response between NGR-TNF andchemotherapy. To assess whether the production of IFN within the tumormicroenvironment could restore the synergistic effect in nu/nu mice, wetransfected TS/A cells with murine IFN cDNA (FIG. 8). One clone able tosecrete IFN in culture medium was selected and named TS/A-IFN. TS/A-IFNand wild-type TS/A cells were then implanted, subcutaneously, in nu/numice. As expected, NGR-TNF/doxorubicin exerted significant anti-tumoreffects against TSA-IFN, but not against TS/A tumors, stronglysuggesting that locally produced IFN is indeed critical for the NGR-TNF/doxorubicin synergism.

[0170] In conclusion, these and the above results indicate that thetherapeutic activity of NGR-TNF/doxorubicin strongly depends on localproduction of IFN, likely secreted by tumor-infiltrating lymphocytes.

[0171] Exogenous IFN Enhances the Therapeutic Activity ofNGR-TNF/Doxorubicin in Immunocompetent Mice.

[0172] The therapeutic activity of NGR-TNF and doxorubicin, incombination with exogenous IFN, (300 ng) was then evaluated using C57B16mice bearing B16F1 tumors. As shown in FIG. 9, the anti-tumor activityof the triple combination was greater than that of NGR-TNF/doxorubicin.Thus, administration of exogenous IFN, together withNGR-TNF/doxorubicin, induced stronger anti-tumor effect also inimmunocompetent mice.

[0173] Mechanism of Action of the Triple Combination (IFN, NGR-TNF andDoxorubicin).

[0174] We have shown previously that an important mechanism for theNGR-TNF/doxorubicin synergism is related to alteration of endothelialbarrier function by NGR-TNF and increased penetration of doxorubicin intumors. Thus, we investigated whether IFN is critical for this effect.To this aim we measured the effect of exogenous IFN on the penetrationof doxorubicin in TS/A tumors (FIG. 10). This experiment takes advantagefrom the fact doxorubicin is a fluorescent compound and that thefluorescence intensity of tumor cells, recovered from animals aftertreatment, is an indication of the amount of doxorubicin that haspenetrated tumors. The experiment was carried out in nu/nu mice toreduce the effect of endogenous IFN. When tumor-bearing mice weretreated with NGR-TNF and, 2 h later, with doxorubicin, no significantincrease of doxorubicin was found in tumor cells, compared to untreatedcontrols. However, administration of IFN in combination with NGR-TNFincreased the penetration of doxorubicin in tumors. This suggests thatIFN is critical for the TNF-induced penetration of chemotherapeuticdrugs.

[0175] The results of this study suggest that endogenous IFN is criticalfor the NGR-TNF/doxorubicin synergistic activity and that exogenousmIFNγ together with NGR-mTNF induce stronger anto-timour effect in bothimmunodeficient and immunocompetent mice. This view is supported by theabove observations that a) a neutralizing anti-IFN antibody markedlyinhibits the NGR-TNF/doxorubicin synergistic activity in immunocompetentmice and b) no synergism occurs in mice lacking the IFN gene (IFN−/−mice).

[0176] Given that T- and NK-cells are the primary sources of IFN inimmunocompetent mice and that athymic nu/nu mice lack T-cells, the aboveresults strongly suggest that T cells are the main source of the IFNnecessary for NGR-TNF/doxorubicin activity. In accord with this view isthe finding that exogenous or endogenous IFN (produced by tumor cellstransfected with IFN cDNA) restore the NGR-TNF/doxorubicin synergisticactivity in nu/nu mice.

[0177] These results point to a crucial role for IFN in tumor vasculartargeting with NGR-TNF and doxorubicin.

[0178] The lack of therapeutic effects of IFN in combination withdoxorubicin (without NGR-TNF) suggest this cytokine synergizes withNGR-TNF, and little or not with doxorubicin. Considering thatchemotherapeutic drugs must cross the vessel wall and migrate throughthe interstitium to reach the cancer cells, the site of action of IFNand NGR-TNF is very likely the endothelial lining of vessels. It is wellknown that TNF can induce, in endothelial cells, alteration ofcytoskeletal actin and formation of intercellular gaps leading toincreased permeability to macromolecules. On the same cells TNF caninduce leukocyte adhesion molecules, proinflammatory cytokines, fibrindeposition, nitric oxide production, and apoptosis. Accordingly, wefound that the in vitro permeability of cA endothelial cell monolayersto horseradish peroxidase was enhanced by exposure to TNF in combinationwith IFN compared to TNF alone. We cannot exclude that, in view of thevarious effects that both TNF and IFN can exert on endothelial cells,alteration of vascular permeability in vivo could be also theconsequence of indirect effects related to local release of otherimportant inflammatory molecules that affect endothelial permeability.

[0179] Our findings may also have other important implications. Severalstudies in animal models and in patients showed that TNF can selectivelyaffect and damage tumor vessels but not vessels associated to normaltissues. Accordingly, the micro- and macrovasculature of tumors, but notof normal tissue, has been observed to be extensively damaged afterpatients were given isolated limb perfusion with TNF in combination withand melphalan. The molecular basis of this selectivity is unclear. Ithas been hypothesized that structural differences within tumor vesselsand/or the presence of tumor derived-“sensitizing factors” could beresponsible for the TNF vascular selectivity. Our results suggest localproduction of IFN, could be one of these sensitizing factors.

[0180] All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are apparent to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

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[0217]37. Moro, M., Pelagi, M., Fulci, G., Paganelli, G., Dellabona, P.,Casorati, G., Siccardi, A. G., and Corti, A. (1997). Cancer Research 57,1922-8.

[0218] 38. Smith, R. A., and Baglioni, C. (1987). Journal of BiologicalChemistry 262, 6951-4.

1 7 1 30 DNA Artificial Sequence Description of Artificial Sequence PCRprimer 1 ctggatcctc acagagcaat gactccaaag 30 2 29 DNA ArtificialSequence Description of Artificial Sequence PCR primer 2 tgcctcacatatgctcagat catcttctc 29 3 46 DNA Artificial Sequence Description ofArtificial Sequence PCR primer 3 gcagatcata tgtgcaacgg ccgttgcggcctcagatcat cttctc 46 4 45 DNA Artificial Sequence Description ofArtificial Sequence PCR primer 4 atatcatatg tgcaacggcc gttgcggcgtcagatcatct tctcg 45 5 36 DNA Artificial Sequence Description ofArtificial Sequence PCR primer 5 tcaggatcct cacagggcaa tgatcccaaa gtagac36 6 35 DNA Artificial Sequence Description of Artificial Sequence PCRprimer 6 atatctacat atgcacggca cagtcattga aagcc 35 7 58 DNA ArtificialSequence Description of Artificial Sequence PCR primer 7 tcggatcctcagcaacggcc gttgcagccg gagcgactcc ttttccgctt cttgaggc 58

1. A conjugation product between a cytokine selected from TNF or IFNγand a ligand of the CD13 receptor.
 2. A conjugation product as claimedin claim 1, wherein said cytokine is TNFγ or TNFβ.
 3. A conjugationproduct as claimed in claim 1, wherein the ligand of the CD13 receptoris selected from the group consisting of antibodies or active fragmentsthereof, peptides or peptido-mimetics.
 4. A conjugation product asclaimed in claim 3, wherein said ligand is a peptide containing the NGRmotif.
 5. A conjugation product as claimed in claim 4, wherein saidpeptide is selected from the group consisting of CNGRCVSGCAGRC, NGRAHA,GNGRG, cycloCVLNGRMEC, linear CNGRC, and cyclic CNGRC.
 6. A conjugationproduct as claimed in claim 1, wherein the cytokine is derivatized withpolyethylene glycol or an acyl residue.
 7. A conjugation product asclaimed in claim 1, wherein the cytokine is further conjugated with acompound selected from the group consisting of an antibody, an antibodyfragment, and biotin, wherein said antibody or fragment thereof isdirected to a compound selected from the group consisting of a tumoralantigen, a tumoral angiogenic marker or a component of the extracellularmatrix.
 8. A conjugation product according to claim 7, wherein thecytokine is TNF and is conjugated to both a CD13 ligand and a compoundselected from the group consisting of an antibody, and antibodyfragment, and biotin.
 9. A cDNA encoding for a cytokine selected fromTNF and IFNγ bearing a 5′ or 3′ contiguous sequence encoding a CD13ligand.
 10. A cDNA according to claim 9, wherein said CD13 ligand is apeptide selected from the group consisting of CNGRCVSGCAGRC, NGRAHA,GNGRG, cycloCVLNGRMEC, linear CNGRC, and cyclic CNGRC.
 11. A vector forgene therapy containing the cDNA of claim
 9. 12. A pharmaceuticalcomposition comprising an effective amount of a conjugation product asclaimed in claim 1, together with pharmaceutically acceptable carriersand excipients.
 13. A composition as claimed in claim 12, in the form ofan injectable solution or suspension or a liquid for infusions.
 14. Acomposition as claimed in claim 12, in the form of liposomes.
 15. Apharmaceutical composition comprising an effective amount of aconjugation product of TNF and a ligand of the CD13 receptor or apolynucleotide encoding therefor, and an effective amount of IFNγ or apolynucleotide encoding therefor.
 16. A composition according to claim15 together with pharmaceutically acceptable carriers and excipients.17. A composition as claimed in claim 15, wherein said TNF is TNFα orTNFβ.
 18. A composition as claimed in claim 15, wherein the ligand ofthe CD13 receptor is selected from the group consisting of antibodies oractive fragments thereof, peptides or peptido-mimetics.
 19. Acomposition as claimed in claim 18, wherein said ligand is a peptidecontaining the NGR motif.
 20. A composition as claimed in claim 19,wherein said peptide is selected from the group consisting ofCNGRCVSGCAGRC, NGRAHA, GNGRG, cycloCVLNGRMEC, linear CNGRC, and cyclicCNGRC.
 21. A composition as claimed in claim 15, wherein the TNF isderivatized with polyethylene glycol or an acyl residue.
 22. Acomposition as claimed in claim 15, wherein the TNF is furtherconjugated with a compound selected from the group consisting of anantibody, an antibody fragment, and biotin, wherein said antibody orfragment thereof is directed to a compound selected from the groupconsisting of a tumoral antigen, a tumoral angiogenic marker or acomponent of the extracellular matrix.
 23. A composition according toclaim 22, wherein the TNF is conjugated to both a CD13 ligand and acompound selected from the group consisting of an antibody, and antibodyfragment, and biotin.
 24. A composition as claimed in claim 15, in theform of an injectable solution or suspension or a liquid for infusions.25. A composition as claimed in claim 15, in the form of liposomes. 26.A composition as claimed in claim 15, further comprising anotherantitumor agent or a diagnostic tumor-imaging compound.
 27. Acomposition as claimed in claim 26, wherein the another antitumor agentis doxorubicin.
 28. A method of treating or diagnosing a cancer patientcomprising administering the conjugation product of claim
 1. 29. Amethod of treating or diagnosing a cancer patient comprisingadministering the cDNA of claim
 9. 30. A method of treating ordiagnosing a cancer patient comprising administering the vector of claim11.
 31. A method of treating or diagnosing a cancer patient comprisingadministering the pharmaceutical composition of claim
 12. 32. A methodof treating or diagnosing a cancer patient comprising administering thepharmaceutical composition of claim
 15. 33. The method of claim 28comprising additionally administering other antitumor agents ordiagnostic tumor-imaging compounds.
 34. The method of claim 29comprising additionally administering other antitumor agents ordiagnostic tumor-imaging compounds.
 35. The method of claim 30comprising additionally administering other antitumor agents ordiagnostic tumor-imaging compounds.
 36. The method of claim 31comprising additionally administering other antitumor agents ordiagnostic tumor-imaging compounds.
 37. The method of claim 32comprising additionally administering other antitumor agents ordiagnostic tumor-imaging compounds.